Towards a molecular understanding of phosphomimetic substitutions.
Lead Research Organisation:
University College London
Department Name: Structural Molecular Biology
Abstract
Strategic Research Priority: World Class Underpinning Bioscience
Abstract
Phosphomimetics are amino acid substitutions that activate (or deactivate) the protein by mimicking a phosphorylated residue. Phosphomimetic mutations are increasingly used to study the effect of phosphorylation and protein activation in vitro and in vivo. This approach however is not always successful as it requires the substituting group and the interactions to have similar features as those of phosphate. No rationale is currently available. The aim of this PhD project is to study by a combination of simulations, NMR and biophysical experiments when and how the approach is feasible, providing a valuable tool for cell and molecular biologists.
Project
Two systems of great biological relevance will be analysed in detail: the interaction between the C-terminus of the ataxin-1 protein and the protein 14-3-3 which is known to depend on the state of phosphorylation of Ser771 of ataxin-1, and the phosphorylation of the activation loop of cSrc. Enhanced sampling molecular dynamics simulations (such as parallel tempering or Metadynamics) will be used to compute the conformational free energy landscape (FES) associated with the activation of the two systems.
The FES of wild-type phosphorylated proteins will be carefully compared to those of the phosphomimetic protein.
The biological activity of the WT, phosphorylated and phosphomimetic proteins with different substitutions will be measured in vitro.
Advanced protein NMR techniques, including relaxation dispersion and RDC together with H/D exchange mass spectrometry will be used to validate and refine the computational results. A significant enabling preliminary work has been already carried out as the NMR spectra of both systems have been already assigned in the labs of the supervisor (cSrc) and co-supervisor
Abstract
Phosphomimetics are amino acid substitutions that activate (or deactivate) the protein by mimicking a phosphorylated residue. Phosphomimetic mutations are increasingly used to study the effect of phosphorylation and protein activation in vitro and in vivo. This approach however is not always successful as it requires the substituting group and the interactions to have similar features as those of phosphate. No rationale is currently available. The aim of this PhD project is to study by a combination of simulations, NMR and biophysical experiments when and how the approach is feasible, providing a valuable tool for cell and molecular biologists.
Project
Two systems of great biological relevance will be analysed in detail: the interaction between the C-terminus of the ataxin-1 protein and the protein 14-3-3 which is known to depend on the state of phosphorylation of Ser771 of ataxin-1, and the phosphorylation of the activation loop of cSrc. Enhanced sampling molecular dynamics simulations (such as parallel tempering or Metadynamics) will be used to compute the conformational free energy landscape (FES) associated with the activation of the two systems.
The FES of wild-type phosphorylated proteins will be carefully compared to those of the phosphomimetic protein.
The biological activity of the WT, phosphorylated and phosphomimetic proteins with different substitutions will be measured in vitro.
Advanced protein NMR techniques, including relaxation dispersion and RDC together with H/D exchange mass spectrometry will be used to validate and refine the computational results. A significant enabling preliminary work has been already carried out as the NMR spectra of both systems have been already assigned in the labs of the supervisor (cSrc) and co-supervisor
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
| 1618890 | Studentship | BB/M009513/1 | 01/10/2015 | 30/09/2019 | Havva Yalinca |